P691: MULTIOMIC SINGLE-CELL APPROACH REVEALED CLONAL ARCHITECTURE OF KIT MUTATIONS IN CHRONIC MYELOMONOCYTIC LEUKAEMIA PATIENTS WITH/WITHOUT SYSTEMIC MASTOCYTOSIS
Authors: Yu-Hung Wang, Kristian Gurashi, Tim C. P. Somervaille, Kiran Batta, Daniel Wiseman
Journal: HemaSphere (2023)

Background: Systemic mastocytosis (SM) involves neoplastic expansion of mast cells associated with activating KIT-D816V mutations. Most Advanced SM patients harbor another hematological neoplasm (“SM-AHN”): often chronic myelomonocytic leukaemia (SM-CMML). Monocytes in SM-CMML can be KIT mutated (KITMT), as can hematopoietic stem cells (HSCs), confirming KITMT as not restricted to the mast cell lineage. Conversely, >5% of CMMLs harbor KIT mutations without overt SM. Treatment for SM targets KITMT with tyrosine kinase inhibitors. However, the relative clonal placement of KITMT, how this contrasts with KITMT in other CMMLs, and longitudinal dynamics of competing clones remain unknown. Aims: We investigated the clonal architecture and hierarchical differentiation landscape of KIT mutations in CMML, with/without concurrent SM, through disease evolution and response to therapy. Methods: Ten bone marrow (BM) samples from 8 KITMT CMML patients were processed: (a) SM-CMML, n=4; (b) non-SM KITMT CMML (“KIT-CMML”), n=3; and (c) SM-CMML post-treatment, n=3 (Fig 1A). BM mononuclear cells were genotyped on the single cell (sc) Tapestri platform, using a custom DNA panel (263 amplicons) and antibody cocktail against 42 surface proteins. Mutations were called, clones resolved and cells annotated using a novel custom pipeline. Results: In total 42,061 cells were genotyped. Median 5 clones and 4 mutations were detected per sample. Median age of SM-CMML was higher than KIT-CMML (76 v 60y, p=0.057). SM-CMML was borderline associated with fewer Ras-pathway mutations (p=0.045) and more transcription factor-mutated clones (p=0.052). All cases harbored a solitary KITMT clone. Interestingly, all KITMT occurred alongside concurrent, pre-existing “CHIP” mutations (most commonly TET2). Number of concurrent mutations in KITMT clones was higher in SM-CMML (median 3 v 2, p=0.036). The fraction of KITMT cells was lower in SM-CMML than KIT-CMML (median 5.4% v 50.7%, p=0.029). KIT mutations were subclonal, in a minority clone, in all SM-CMML samples. By contrast, in every KIT-CMML case the KIT mutation occurred in the dominant (or near-dominant) clone. Among 19 populations separated immunophenotypically (Fig 1B), as expected SM-CMML carried higher mast cell proportions (median 2.8% v 0.9%, p=0.002). Interestingly, the proportion of KITMT mast cells was similar in SM-CMML and KIT-CMML. Meanwhile, the proportion of KITMT HSCs was markedly higher in KIT-CMML (median 58.6% v 0.6%, p=0.008), consistent with origination in a more apical HSC with multilineage differentiation potential. Post-treatment samples all displayed suppression (or loss) of KITMT clones, but marked expansion of clones carrying epigenetic (ASXL1/IDH1/IDH2) and/or signalling (NRAS) genes (Fig 1C): commensurate with observed patterns of non-mast cell disease coming to dominate the clinical picture. Lymphoid lineages were enriched post-treatment, while primitive fractions were also suppressed. Conclusion: We characterize the clonal architecture of KITMT-CMML with/without SM, noting distinct patterns of KITMT origination and propagation. KIT mutations were always detected in apical HSCs, accompanied by CHIP mutations: contrasting with sporadic SM, in which KIT is typically the sole mutation. The fraction of KITMT cells was much lower in SM-CMML, indicating SM as a subclonal branch of a broader disease complex. Further studies are needed to decipher the cell-of-origin, pathogenesis trajectories and clonal relationships within SM-AHN. However, we show progression clearly driven by KIT-WTwild type clones, highlighting the importance of therapeutic targeting beyond the KITMT clone and potential for serial sc genotyping to personalize adaptive therapy. Figure 1.Keywords: Kit, Systemic mastocytosis, CMML, Clonal expansion